330 PATHOGENIC MICROORGANISMS 



quently able to show that the substance causing the death and partial 

 solution of the leucocytes was a soluble toxin formed by the staphylo- 

 coccus, not only in vivo, but in vitro as well; for cultures of Staphylo- 

 coccus pyogenes aureus, grown in mixtures of bouillon and blood 

 serum, contained, within forty-eight hours, marked quantities of this 

 "leucocidin." 



Other workers since Van de Velde have evolved various methods for 

 obtaining potent leucocidin. Bail 1 obtained it by growing virulent 

 staphylococcus in mixtures of one-per-cent glycerin solutions and rab- 

 bit serum. Neisser and Wechsberg 2 advise the use of a carefully titrated 

 alkaline bouillon. To obtain the leucocidin ^ee from bacteria, the 

 cultures are passed through Chamberland or Berkefeld filters, after 

 about eight to eleven days' growth at 37 C., at which time the con- 

 tents in leucocidin are usually at their highest point. 



The action of leucocidin upon leucocytes may be observed in vivo 

 by the simple method of Van de Velde, of injecting virulent staphylo- 

 cocci intrapleurally into rabbits and examining the exudate. Bail 

 advises the production of leucocytic intrapleural exudates by the use 

 of aleuronat and following this after twenty-four hours by an injection 

 of leucocidin-filtrate. In vitro the phenomenon may be observed by 

 direct examination of mixtures of leucocytes and leucocidin in the 

 hanging drop on a warmed stage, or by the " methylene-blue method", 

 of Neisser and Wechsberg. This method is based upon the fact that 

 living leucocytes will reduce methylene-blue solutions and render them 

 colorless, while dead leucocytes have lost this power. Leucocidin and 

 leucocytes are allowed to remain in contact for a given time and to them 

 is then added an extremely dilute solution of methylene-blue. If the 

 leucocytes have been actively attacked by- leucocidin, no reduction takes 

 place. This method is particularly adapted for quantitative tests. 



All staphylococcus strains do not produce leucocidin to the same 

 degree. Almost all true Staphylococcus pyogenes aureus cultures 

 produce some of this toxin, but one strain may produce fifty- and a 

 hundred-fold the quantity produced by another. Staphylococcus 

 pyogenes albus gives rise to this substance but rarely, and then in small 

 quantity. 



Leucocidin seems to be similar to the soluble toxins of other bacteria. 

 It is rapidly destroyed by heat at 58 C., and deteriorates quickly in 



*Bail, Arch. f. Hyg., xxxii, 1898. 



8 Neisser und Wechsberg, Zeit. f. Hyg., xxxvi, 1901. 



